Van Hove Bob, Guidi Chiara, De Wannemaeker Lien, Maertens Jo, De Mey Marjan
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University , 9000 Ghent, Belgium.
ACS Synth Biol. 2017 Jun 16;6(6):943-949. doi: 10.1021/acssynbio.7b00017. Epub 2017 Mar 24.
A problem rarely tackled by current DNA assembly methods is the issue of cloning additional parts into an already assembled construct. Costly PCR workflows are often hindered by repeated sequences, and restriction based strategies impose design constraints for each enzyme used. Here we present Protected Oligonucleotide Duplex Assisted Cloning (PODAC), a novel technique that makes use of an oligonucleotide duplex for iterative Golden Gate cloning using only one restriction enzyme. Methylated bases confer protection from digestion during the assembly reaction and are removed during replication in vivo, unveiling a new cloning site in the process. We used this method to efficiently and accurately assemble a biosynthetic pathway and demonstrated its robustness toward sequence repeats by constructing artificial CRISPR arrays. As PODAC is readily amenable to standardization, it would make a useful addition to the synthetic biology toolkit.
当前DNA组装方法很少解决的一个问题是将额外的片段克隆到已组装好的构建体中的问题。昂贵的PCR工作流程常常受到重复序列的阻碍，而基于限制性内切酶的策略对每种使用的酶都施加了设计限制。在此，我们介绍了受保护的寡核苷酸双链体辅助克隆（PODAC），这是一种新技术，它利用寡核苷酸双链体仅使用一种限制性内切酶进行迭代金门克隆。甲基化碱基在组装反应期间赋予免受消化的保护，并在体内复制期间被去除，在此过程中揭示一个新的克隆位点。我们使用这种方法高效且准确地组装了一条生物合成途径，并通过构建人工CRISPR阵列证明了其对序列重复的稳健性。由于PODAC易于标准化，它将成为合成生物学工具包的一个有用补充。
